Glycosylated Porphyra-334 and Palythine-Threonine from the Terrestrial Cyanobacterium Nostoc commune

Mycosporine-like amino acids (MAAs) are water-soluble UV-absorbing pigments, and structurally different MAAs have been identified in eukaryotic algae and cyanobacteria. In this study novel glycosylated MAAs were found in the terrestrial cyanobacterium Nostoc commune (N. commune). An MAA with an absorption maximum at 334 nm was identified as a hexose-bound porphyra-334 derivative with a molecular mass of 508 Da. Another MAA with an absorption maximum at 322 nm was identified as a two hexose-bound palythine-threonine derivative with a molecular mass of 612 Da. These purified MAAs have radical scavenging activities in vitro, which suggests multifunctional roles as sunscreens and antioxidants. The 612-Da MAA accounted for approximately 60% of the total MAAs and contributed approximately 20% of the total radical scavenging activities in a water extract, indicating that it is the major water-soluble UV-protectant and radical scavenger component. The hexose-bound porphyra-334 derivative and the glycosylated palythine-threonine derivatives were found in a specific genotype of N. commune, suggesting that glycosylated MAA patterns could be a chemotaxonomic marker for the characterization of the morphologically indistinguishable N. commune. The glycosylation of porphyra-334 and palythine-threonine in N. commune suggests a unique adaptation for terrestrial environments that are drastically fluctuating in comparison to stable aquatic environments.     

Analysis of Mycosporine-Like Amino Acids in Selected Algae and Cyanobacteria by Hydrophilic Interaction Liquid Chromatography and a Novel MAA from the Red Alga Catenella repens

Mycosporine-like amino acids (MAAs), a group of small secondary metabolites found in algae, cyanobacteria, lichens and fungi, have become ecologically and pharmacologically relevant because of their pronounced UV-absorbing and photo-protective potential. Their analytical characterization is generally achieved by reversed phase HPLC and the compounds are often quantified based on molar extinction coefficients. As an alternative approach, in our study a fully validated hydrophilic interaction liquid chromatography (HILIC) method is presented. It enables the precise quantification of several analytes with adequate retention times in a single run, and can be coupled directly to MS. Excellent linear correlation coefficients (R² > 0.9991) were obtained, with limit of detection (LOD) values ranging from 0.16 to 0.43 µg/mL. Furthermore, the assay was found to be accurate (recovery rates from 89.8% to 104.1%) and precise (intra-day precision: 5.6%, inter-day precision ≤6.6%). Several algae were assayed for their content of known MAAs like porphyra-334, shinorine, and palythine. Liquid chromatography-mass spectrometry (LC-MS) data indicated a novel compound in some of them, which could be isolated from the marine species Catenella repens and structurally elucidated by nuclear magnetic resonance spectroscopy (NMR) as (E)-3-hydroxy-2-((5-hydroxy-5-(hydroxymethyl)-2-methoxy-3-((2-sulfoethyl)amino)cyclohex-2-en-1-ylidene)amino) propanoic acid, a novel MAA called catenelline.     

Status of Bactrocera invadens (Diptera: Tephritidae) in Mango-Producing Areas of Arba Minch,Southwestern Ethiopia

Bactrocera invadens, the Asian fruit fly, was first reported in Kenya in 2003, and it spread fast to most tropical countries in Africa. To our knowledge, there is no detailed data on the fruit damage and status of fruit flies in Arba Minch and elsewhere in Ethiopia. Hence, information on the species composition and pest status of the fruit fly species is urgent to plan management strategies in the area. Fruit flies were captured using male parapheromone-baited traps. Matured mango (Mangifera indica) fruits were collected from randomly selected mango trees and incubated individually in cages (15 by 15 by 15 cm) with sandy soil. B. invadens was the predominant (96%; 952 of 992) captured species and the only fruit fly species emerging from mango fruits incubated in the laboratory. The mean number of adult B. invadens emerging per mango fruit was 35.25, indicating that the species is the most devastating mango fruit fly in the area. The loss due to this species would be serious if no management strategies are implemented.     

Valorization of the Red Algae Gelidium sesquipedale by Extracting a Broad Spectrum of Minor Compounds Using Green Approaches

Until now, the red algae Gelidium sesquipedale has been primarily exploited for agar production, leaving an undervalued biomass. In this work, the use of eco-friendly approaches employing ultrasound-assisted extraction (UAE) and green solvents was investigated to valorize the algal minor compounds. The green methods used herein showed an attractive alternative to efficiently extract a broad spectrum of bioactive compounds in short extraction times (15 to 30 min vs. 8 h of the conventional method). Using the best UAE conditions, red seaweed extracts were characterized in terms of total phenolics (189.3 ± 11.7 mg GAE/100 g dw), flavonoids (310.7 ± 9.7 mg QE/100 g dw), mycosporine-like amino acids (MAAs) (Σ MAAs = 1271 mg/100 g dw), and phycobiliproteins (72.4 ± 0.5 mg/100 g dw). Additionally, produced algal extracts exhibited interesting antioxidant and anti-enzymatic activities for potential applications in medical and/or cosmetic products. Thus, this study provides the basis to reach a superior valorization of algal biomass by using alternative methods to extract biologically active compounds following eco-friendly approaches. Moreover, the strategies developed not only open new possibilities for the commercial use of Gelidium sesquipedale, but also for the valorization of different algae species since the techniques established can be easily adapted.     

Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.     

Pigment Composition of Nine Brown Algae from the Iberian Northwestern Coastline: Influence of the Extraction Solvent

Brown algae are ubiquitously distributed in the NW coastline of the Iberian Peninsula, where they stand as an underexploited resource. In this study, five solvents were applied to the extraction of pigments from nine brown algae, followed by their determination and quantification by HPLC-DAD. A total of 13 compounds were detected: Six were identified as chlorophylls, six were classified as xanthophylls, and one compound was reported as a carotene. Fucoxanthin was reported in all extracts, which is the most prominent pigment of these algae. Among them, L. saccharina and U. pinnatifida present the highest concentration of fucoxanthin (4.5–4.7 mg∙g⁻¹ dry weight). Ethanol and acetone were revealed as the most efficient solvents for the extraction of pigments, showing a maximal value of 11.9 mg of total pigments per gram of dry alga obtained from the ethanolic extracts of H. elongata, followed by the acetonic extracts of L. ochroleuca. Indeed, ethanol was also revealed as the most efficient solvent according to its high extraction yield along all species evaluated. Our results supply insights into the pigment composition of brown algae, opening new perspectives on their commercial exploitation by food, pharmaceutical, and cosmeceutical industries.     

Natural Products from Antarctic Colonial Ascidians of the Genera Aplidium and Synoicum: Variability and Defensive Role

Ascidians have developed multiple defensive strategies mostly related to physical, nutritional or chemical properties of the tunic. One of such is chemical defense based on secondary metabolites. We analyzed a series of colonial Antarctic ascidians from deep-water collections belonging to the genera Aplidium and Synoicum to evaluate the incidence of organic deterrents and their variability. The ether fractions from 15 samples including specimens of the species A. falklandicum, A. fuegiense, A. meridianum, A. millari and S. adareanum were subjected to feeding assays towards two relevant sympatric predators: the starfish Odontaster validus, and the amphipod Cheirimedon femoratus. All samples revealed repellency. Nonetheless, some colonies concentrated defensive chemicals in internal body-regions rather than in the tunic. Four ascidian-derived meroterpenoids, rossinones B and the three derivatives 2,3-epoxy-rossinone B, 3-epi-rossinone B, 5,6-epoxy-rossinone B, and the indole alkaloids meridianins A–G, along with other minoritary meridianin compounds were isolated from several samples. Some purified metabolites were tested in feeding assays exhibiting potent unpalatabilities, thus revealing their role in predation avoidance. Ascidian extracts and purified compound-fractions were further assessed in antibacterial tests against a marine Antarctic bacterium. Only the meridianins showed inhibition activity, demonstrating a multifunctional defensive role. According to their occurrence in nature and within our colonial specimens, the possible origin of both types of metabolites is discussed.     

Connection between primary Fusarium inoculum on gramineous weeds, crop residues and soil samples and the final population on wheat ears in Flanders, Belgium

Fusarium head blight (FHB) is a devastating wheat disease and is influenced by weather conditions and agronomic factors. Since FHB is a mostly monocyclic disease, the quantity of primary inoculum is a key factor influencing its incidence. To investigate the connection between the primary Fusarium inoculum and the final population on wheat ears, naturally occurring populations of Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium poae and Microdochium nivale were studied at eight locations in Flanders, Belgium during the growing seasons of 2008-2009 and 2009-2010. To determine the composition of the primary inoculums in November, weeds, wheat and maize residues as well as soil samples were examined. At the end of the growing season, in July, the population on wheat ears was determined. In both growing seasons, the population was characterized by a large complexity and a differential composition at each location and for each type of sample. Nevertheless, some clear correspondences were observed: F. culmorum was a predominant species in crop residues and in soil samples in November, while the population on wheat ears in July consisted mainly of F. graminearum and F. poae, with only a lower frequency of F. culmorum, indicating that soil is not an important source of primary inoculum. The presence of M. nivale was restricted to weeds, crop residues and soil samples in November, and was nearly absent in July at the majority of locations. Finally, our results also indicate that the Fusarium population in July is more complex than the population at the beginning of the season in November. The information of the primary inoculum and the composition of the FHB population at the end of the growing season is important to predict FHB incidence and to implement control strategies for FHB.     

Usefulness of Japanese-radish residue in biological soil disinfestation to suppress spinach wilt disease accompanying with proliferation of soil bacteria in the Firmicutes

Biological soil disinfestation (BSD) is an effective method to suppress soilborne plant diseases by incorporation of plant biomass into soil under reduced, anoxic condition. Usefulness of Japanese-radish (daikon) residue as plant biomass for BSD was investigated by both model and field experiments in comparison with the effects of Brassica juncea plants or wheat bran. Considerable amounts of acetate together with minor amounts of propionate and butyrate were detected from the radish-treated soils at similar levels with those in soils treated with B. juncea plants or wheat bran. BSD treatments with radish residue reduced spinach wilt disease incidence in both model and field experiments. When the BSD-treated soil was treated again with irrigation and covering without biomass before next cropping, however, wilt disease was hardly suppressed. Clone library analysis based on 16S rRNA gene sequences was carried out to determine the changes in the bacterial community compositions in the treated soil samples. The analyses showed that the bacterial communities in the radish-treated soils were dominated by members of the classes Clostridia and Bacilli of the phylum Firmicutes in both experiments. The clostridial groups detected were diverse and the major operational taxonomic units (OTUs) were closely related to Clostridium saccharobutylicum, Clostridium sufflavum, Clostridium xylanovorans, and Oxobacter pfennigii, which had been commonly detected as the dominant groups in BSD-soils treated with B. juncea plants or wheat bran in our previous studies. The dominant clone groups belonging to the Bacilli class were closely related to several species such as Bacillus niacini, Bacillus circulans, and Bacillus pycnus. Dominancy of the Bacilli groups seemed to increase when radish residue was repeatedly applied as BSD material.     

Comparative chemistry and insect antifeedant action of traditional (Clevenger and Soxhlet) and supercritical extracts (CO2) of two cultivated wormwood (Artemisia absinthium L.) populations

A comparison between traditional extraction techniques (hydrodistillation and organic solvent extraction) and supercritical fluid extraction was made for two different populations and crops of Artemisia absinthium L., cultivated in the field and aeroponically. The composition of the extracts, volatile and non volatile oils, was analyzed by GC-MS and HPLC-DAD, respectively. The antifeedant and phytotoxic activity of the extracts was tested on insect pests (Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi) and plants (Lactuca sativa and Lolium perenne). The supercritical extracts exhibited stronger antifeedant effects than the traditional ones (up to 8 times more active) with moderate selective phytotoxic effects on L. perenne root growth (<50% inhibition).     

Efficient Isolation of Mycosporine-Like Amino Acids from Marine Red Algae by Fast Centrifugal Partition Chromatography

Marine rhodophyta are known to synthesize specific secondary metabolites, mycosporine-like amino acids (MAAs), to protect themselves from harmful UV-radiation. Shinorine and porphyra-334 are among the most abundant representatives of this compound class. In the present work, a novel approach for their isolation is described. As a first step, a fast centrifugal partition chromatography method, with an aqueous two-phase system comprising water, ethanol, ammonium sulfate and methanol in ascending mode, was developed to isolate the two MAAs from crude aqueous-methanolic extracts of three algal species within 90 min. The compounds could be isolated when just one of them was present in a sample or also both at the same time. By employing solid phase extraction as a second purification step, the individual MAAs were obtained in high purity and good quantity within a much shorter time frame than the established purification protocols, e.g., semi-preparative HPLC. For example, from 4 g Porphyra sp. (Nori) crude extract, 15.7 mg shinorine and 36.2 mg porphyra-334 were isolated. Both were highly pure, as confirmed by TLC, HPLC-MS and NMR analyses.     

The Effect of Dissolved Polyunsaturated Aldehydes on Microzooplankton Growth Rates in the Chesapeake Bay and Atlantic Coastal Waters

Allelopathy is wide spread among marine phytoplankton, including diatoms, which can produce cytotoxic secondary metabolites such as polyunsaturated aldehydes (PUA). Most studies on diatom-produced PUA have been dedicated to their inhibitory effects on reproduction and development of marine invertebrates. However, little information exists on their impact on key herbivores in the ocean, microzooplankton. This study examined the effects of dissolved 2E,4E-octadienal and 2E,4E-heptadienal on the growth rates of natural ciliate and dinoflagellate populations in the Chesapeake Bay and the coastal Atlantic waters. The overall effect of PUA on microzooplankton growth was negative, especially at the higher concentrations, but there were pronounced differences in response among common planktonic species. For example, the growth of Codonella sp., Leegaardiella sol, Prorodon sp., and Gyrodinium spirale was impaired at 2 nM, whereas Strombidium conicum, Cyclotrichium gigas, and Gymnodinium sp. were not affected even at 20 nM. These results indicate that PUA can induce changes in microzooplankton dynamics and species composition.     

Mycosporine-Like Amino Acids from Marine Resource

In the last 10 years, a great number of publications (both regular papers and reviews) have been published on the interesting molecules—mycosporine-like amino acids (MAAs). Despite significant advances in the research of MAAs, current overviews in the recent publications involving MAA research still need reporting. The aim of this Special Issue is to join, as an interdisciplinary approach, the photochemical and photobiological aspects, with emphasis on new natural resources to obtain both algae and zooplankton MAAs, advances in methodology of extraction and chemical identification of new MAAs. Finally, this Special Issue reviews the bioactivities of MAAs including UVR screen, antioxidant, immunostimulant, growth factor, DNA protection, inhibition of collagenase, elastase and hyaluronidase, and anti-photoaging, among others, and their potential use as nutracosmeceutic molecules (i.e., oral and topic photoprotector).     

Characterization of Antioxidant Activity of Heated Mycosporine-like Amino Acids from Red Alga Dulse Palmaria palmata in Japan

We recently demonstrated the monthly variation and antioxidant activity of mycosporine-like amino acids (MAAs) from red alga dulse in Japan. The antioxidant activity of MAAs in acidic conditions was low compared to that in neutral and alkali conditions, but we found strong antioxidant activity from the heated crude MAA fraction in acidic conditions. In this study, we identified and characterized the key compounds involved in the antioxidant activity of this fraction. We first isolated two MAAs, palythine, and porphyra-334, from the fraction and evaluated the activities of the two MAAs when heated. MAAs possess absorption maxima at around 330 nm, while the heated MAAs lost this absorption. The heated MAAs showed a high ABTS radical scavenging activity at pH 5.8–8.0. We then determined the structure of heated palythine via ESI-MS and NMR analyses and speculated about the putative antioxidant mechanism. Finally, a suitable production condition of the heated compounds was determined at 120 °C for 30 min at pH 8.0. We revealed compounds from red algae with antioxidant activities at a wide range of pH values, and this information will be useful for the functional processing of food.     

Identification of Rosellinia species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus Dematophora as inferred from polythetic taxonomy

Rosellinia (Xylariaceae) is a large, cosmopolitan genus comprising over 130 species that have been defined based mainly on the morphology of their sexual morphs. The genus comprises both lignicolous and saprotrophic species that are frequently isolated as endophytes from healthy host plants, and important plant pathogens. In order to evaluate the utility of molecular phylogeny and secondary metabolite profiling to achieve a better basis for their classification, a set of strains was selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, beta-tubulin (TUB2) and the second largest subunit of the RNA polymerase II (RPB2). Concurrently, various strains were surveyed for production of secondary metabolites. Metabolite profiling relied on methods with high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS) as well as preparative isolation of the major components after re-fermentation followed by structure elucidation using nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). Two new and nine known isopimarane diterpenoids were identified during our mycochemical studies of two selected Dematophora strains and the metabolites were tested for biological activity. In addition, the nematicidal cyclodepsipeptide PF1022 A was purified and identified from a culture of Rosellinia corticium, which is the first time that this endophyte-derived drug precursor has been identified unambiguously from an ascospore-derived isolate of a Rosellinia species. While the results of this first HPLC profiling were largely inconclusive regarding the utility of secondary metabolites as genus-specific chemotaxonomic markers, the phylogeny clearly showed that species featuring a dematophora-like asexual morph were included in a well-defined clade, for which the genus Dematophora is resurrected. Dematophora now comprises all previously known important plant pathogens in the genus such as D. arcuata, D. bunodes, D. necatrix and D. pepo, while Rosellinia s. str. comprises those species that are known to have a geniculosporium-like or nodulisporium-like asexual morph, or where the asexual morph remains unknown. The extensive morphological studies of L.E. Petrini served as a basis to transfer several further species from Rosellinia to Dematophora, based on the morphology of their asexual morphs. However, most species of Rosellinia and allies still need to be recollected in fresh state, cultured, and studied for their morphology and their phylogenetic affinities before the infrageneric relationships can be clarified.     

Rapid and Accurate Identification by Real-Time PCR of Biotoxin-Producing Dinoflagellates from the Family Gymnodiniaceae

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.     

Correlation of fruit fly (Diptera Tephritidae) infestation of major mango cultivars in Borgou (Benin) with abiotic and biotic factors and assessment of damage

Fruit flies associated with mango trees were monitored in two orchards in Benin using traps baited with methyl eugenol, terpinyl acetate and Torula during 2005–2006. Population fluctuations were analysed with respect to environmental factors including air temperature, relative humidity and rainfall in relation to different mango cultivars. Mangoes were sampled every two weeks during the two crop years, to assess the damage caused by these quarantine pests on ten main cultivars. Three native species of Ceratitis and a recently described new exotic species, Bactrocera invadens made up the complex of economically significant fruit flies associated with the mango tree in Borgou. Ceratitis species occurred during the dry season and the main species, Ceratitis cosyra, reached a peak at the end of the dry season. B. invadens populations were scarce during the dry season, but increased steadily from the end of April to reach a peak at the end of June during the rainy season. Regression analyses indicated that minimum–maximum temperature, relative humidity and rainfall were the major climatic factors influencing fly populations. Daily rainfall was the factor showing the strongest positive correlation with B. invadens populations. Host plant was another essential factor influencing the population fluctuations. Trapping and rearing data indicated that Ceratitis quinaria and Ceratitis silvestrii, were abundant only in the dry season, causing damage only to early cultivars. C. cosyra, also common during the dry season, attacked both early cultivars or mid season cultivars. A consistent population increase of B. invadens in the early rainy season caused considerable damage to mid season and late cultivars. The seasonal increase of the B. invadens population coincided with the fruiting period of the main mango cultivars in this Northern Guinean savannah, but mango availability influenced the population of this new invasive species only when the rains had arrived. Mean damage on mangoes for the two seasons and two studied orchards increased from 17% in early April to 73% at mid June.     

Complementary UV-Absorption of Mycosporine-like Amino Acids and Scytonemin is Responsible for the UV-Insensitivity of Photosynthesis in Nostoc flagelliforme

Mycosporine-like amino acids (MAAs) and scytonemin are UV-screening compounds that have presumably appeared early in the history of life and are widespread in cyanobacteria. Natural colonies of the UV-insensitive Nostoc flagelliforme were found to be especially rich in MAAs (32.1 mg g DW⁻¹), concentrated in the glycan sheath together with scytonemin. MAAs are present in the form of oligosaccharide-linked molecules. Photosystem II activity, measured using PAM fluorescence and oxygen evolution, was used as a most sensitive physiological parameter to analyse the effectiveness of UV-protection. Laboratory experiments were performed under controlled conditions with a simulated solar radiation specifically deprived of UV-wavebands with cut-off filters (295, 305, 320, 345 and 395 nm). The UV-insensitivity of N. flagelliforme was found to cover the whole UV-A (315–400 nm) and UV-B (280–320 nm) range and is almost certainly due to the complementary UV-absorption of MAAs and scytonemin. The experimental approach used is proposed to be suitable for the comparison of the UV-protection ability in organisms that differ in their complement of UV-sunscreen compounds. Furthermore, this study performed with a genuinely terrestrial organism points to the relevance of marine photoprotective compounds for life on Earth, especially for the colonization of terrestrial environments.     

Transmission Efficiency of <Emphasis Type="Italic">Potato Leafroll Virus</Emphasis> by Four Potato Colonizing Aphid Species in Tunisian Potato Fields

This study investigated the transmission efficiency of Potato leafroll virus (PLRV) by four potato colonizing aphid species, Myzus persicae, Macrosiphum euphorbiae, Aphis gossypii and Aphis fabae, reported from leaves and yellow water trap. Physalis floridana was used as a test plant for virus transmission. DAS-ELISA was used for virus screening of samples as well as virus detection on the test plant after transmission experiment. A 2-h period was sufficient for the tested aphids to acquire PLRV virions. However, a difference in the transmission potential occurred according to the aphid species. The highest potential was recorded for M. persicae and M. euphorbiae, at 90 and 80%, respectively. For the first time, the study revealed the PLRV transmission efficiency of A. fabae, estimated at 50%. The lowest potential rate of 30% was recorded for A. gossypii. This study highlights the PLRV transmission capacities of four potato colonizing aphids suspected to play a key role in the spread of PLRV in potato seed production sites.